Field of the Invention
The present invention relates to the field of medical treatment of diseases and disorders, as well as the field of biomedical engineering. Embodiments of the invention relate to the delivery of Irreversible Electroporation (IRE) through the vasculature of organs to treat tumors embedded deep within the tissue or organ, or to decellularize organs to produce a scaffold from existing animal tissue, such as human tissue, with the existing vasculature intact. In addition, embodiments of the invention may be used in the treatment of malignant and benign diseases through the enhanced administration of therapeutic drugs or gene constructs by facilitating reversible electroporation. Further, vascular electrical conduits may be used to administer other therapies that rely on the delivery of electrical energy to a targeted region of the body or organ tissue with the existing vasculature intact.
Description of Related Art
The ablation of unwanted soft tissue can be achieved by many means, including surgical excision, application of excessive amount of ionizing radiation or other forms of energy (excessive heating and cooling), exposure to cytotoxic chemicals, or by a combination of these means. It is common to use these means to destroy neoplasms. Treatments known in the art involve surgical intervention to physically remove the aberrant cell mass, radiation to kill the cells of the aberrant cell mass, exposure of aberrant cells to toxic chemicals (i.e., chemotherapy), or a combination of such techniques. While each treatment modality has shown significant effectiveness in treatment of various cell proliferative diseases, no one technique has been shown to be highly effective at treating all types of cell proliferative diseases and disorders.
While surgical intervention is effective at removal of solid tumors on tissues and organs that are physically accessible and capable of sustaining physical damage or capable of regeneration, surgical intervention can be difficult to perform on tumors that are not readily accessible or on organs that do not regenerate. In these cases, surgical intervention can often involve substantial physical damage to the patient, requiring extensive recuperation times and follow-on treatments. At other times, the extensive growth of the neoplasm prevents removal, since attempts at removal would likely kill the patient. Likewise, treatment with radiation can result in collateral damage to tissue surrounding the tumor, and can cause long-lasting side-effects, which can lower the quality of life of the patient. Chemotherapeutic treatments can cause systemic damage to the patient, and can result in significant side effects that might require a long recuperation period or cause permanent damage to tissues and organs.
Recent work by the inventors has focused on the ablation of unwanted soft tissue (malignant tumors) by application of excessive electrical energy, using a technique termed Irreversible Electroporation (IRE). Successful control and/or ablation of soft tissue sarcoma and malignant glioma have been achieved. Irreversible electroporation (IRE) involves placing electrodes within or near the targeted region to deliver a series of low energy, microsecond electric pulses. These pulses permanently destabilize the cell membranes of the targeted tissue (e.g., tumor), thereby killing the cells. When applied with precision, IRE does not damage major blood vessels, does not require the use of drugs and non-thermally kills neoplastic cells in a controllable manner, without significantly damaging surrounding tissue.
Other methods of treating disease involve replacing diseased tissue or organs. Over the past twenty years, organ transplantation has become a standard care for patients diagnosed with end stage diseases like cirrhosis, renal failure, etc. The extraordinary success of liver transplantation, with 90% and 75% survival rates after 1 and 5 years, respectively, has led to a progressively increasing number of patients awaiting transplant. Chan, S. C. et al., A decade of right liver adult-to-adult living donor liver transplantation—The recipient mid-term outcomes. Annals of Surgery 248, 411-418, doi:10.1097/SLA.0b013e31818584e6 (2008) (“Chan 2008”).
According to the United Network of Organ Sharing (UNOS), there are over 108,000 candidates in the US alone currently waiting for organ transplants including kidney, liver, heart, lung, and many others. Of those, there are over 16,000 candidates in immediate (one year) need of a liver transplant, and at least 100,000 additional patients with advanced liver disease who would benefit from one. In 2009, there were fewer than 7,000 liver transplants from both living and deceased donors. United Network of Organ Sharing, <http://www.unos.org>(2010).
Standard liver transplantation in the US is usually predicated on organ removal from the donor coincident with the onset of brainstem death. See Kootstra, G., Daemen, J. H. C. & Oomen, A. P. A. Categories of Non-Heart-Beating Donors, Transplant. Proc. 27, 2893-2894 (1995); Rigotti, P. et al. Non-Heart-beating Donors—An Alternative Organ Source in Kidney Transplantation, Transplant. Proc. 23, 2579-2580 (1991); and Balupuri, S. et al. The trouble with kidneys derived from the non heart-beating donor: A single center 10-year experience. Transplantation 69, 842-846 (2000).
Despite advances in transplant surgery and general medicine, the number of patients awaiting transplant organs continues to grow, while the supply of organs does not. The growing discrepancy between organ supply and clinical demand is due to a number of factors including an increase in population age, an increasing incidence of diseases requiring liver transplants (hepatocellular carcinoma and infection with hepatitis viruses), rapid organ degradation (hours) after donation, mismatches created by histocompatibility and other immunologic phenomena, and size mismatches between organs and potential recipients (including pediatric patients), transplantation is the only workable treatment for patients suffering end stage liver disease (Murray, K. F. & Carithers, R. L. AASLD practice guidelines: Evaluation of the patient for liver transplantation. Hepatology 41, 1407-1432, doi:10.1002/hep.20704 (2005)); increased incidence of non-alcoholic fatty liver disease (Amarapurkar, D. N. et al. How common is non-alcoholic fatty liver disease in the Asia-Pacific region and are there local differences? J. Gastroenterol. Hepatol. 22, 788-793, doi:10.1111/j.1440-1746.2007.05042.x (2007)); and acceptance of transplantation for patients with metabolic or congenital diseases (Miro, J. M., Laguno, M., Moreno, A., Rimola, A. & Hosp Clin, O. L. T. H. I. V. W. G. Management of end stage liver disease (ESLD): What is the current role of orthotopic liver transplantation (OLT)? J. Hepatol. 44, S140-S145, doi:10.1016/j.jhep.2005.11.028 (2006)).
Organ supply is constrained by obstacles that impede acquisition. For example, the requirement for organ removal coincident with brainstem death necessitates the use of hospital resources to maintain artificial life support. As a result, organ donation may be problematic when intensive care resources are strained. Fabre. Report of the British Transplantation Society Working Party on Organ Donation. (1995). Use of life support for preservation of potential organ donations has been ethically debated (See Feest, T. G. et al., Protocol for Increasing Organ Donation After Cerebrovascular Deaths in a District General Hospital, Lancet 335, 1133-1135 (1990); and Riad, H. & Nicholls, A., An Ethical Debate Elective Ventilation of Potential Organ Donors, Br. Med. J. 310, 714-715 (1995)), and donation refusal is common in regions where social, cultural, and religious pressures place constraints on organ procurement.
The increasing gap between organ donation and supply to patients has caused an increased interest in alternative organ sources. Perera, M., Mirza, D. F. & Elias, E. Liver transplantation: Issues for the next 20 years. J. Gastroenterol. Hepatol. 24, S124-S131, doi:10.1111/j.1440-1746.2009.06081.x (2009). Developing engineered materials to replicate the structure and function of organs has been met with limited success. Large volumes of poorly-organized cells and tissues cannot be implanted due to the initial limited diffusion of oxygen, nutrients and waste. Folkman, J. Self-Regulation of Growth In 3 Dimensions. Journal of Experimental Medicine 338 (1973); and Kaufman, D. S., Hanson, E. T., Lewis, R. L., Auerbach, R. & Thomson, J. A. Hematopoietic colony-forming cells derived from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 98, 10716-10721 (2001).
Despite this, researchers have made some progress toward partial organ regeneration. For instance, mouse renal cells, grown on decellularized collagen matrices and implanted into athymic mice, developed nephron-like structures after 8 weeks. Atala, A. Engineering organs. Curr. Opin. Biotechnol. 20, 575-592, doi:10.1016/j.copbio.2009.10.003 (2009) (“Atala 2009”). In addition, five millimeter thick porous polyvinyl-alcohol (PVA) constructs, implanted in mice and then injected with hepatocytes, developed liver-like morphology over the course of one year. Kaufmann, P. M. et al. Long-term hepatocyte transplantation using three-dimensional matrices. Transplant. Proc. 31, 1928-1929 (1999) (“Kaufmann 1999”). Cell survival and proliferation in each of these structures was limited to a few millimeters from a nutrient source.
For the development and differentiation of full organs suitable for human transplantation, structures that provide microvasculature needed for the delivery of nutrients throughout the tissue and a physical semirigid matrix for cellular organization and anchorage must be developed. See Atala 2009; Kaufmann 1999; and Atala, A. Experimental and clinical experience with tissue engineering techniques for urethral reconstruction. Urologic Clinics of North America 29, 485-+ (2002). Traditional top-down manufacturing techniques are currently unable to produce a hierarchical vascular structure which, in human organs, ranges in size from a several centimeters (vena cava, for example) down to only a few micrometers (most capillaries), a scale spanning more than four orders of magnitude. Microfabrication techniques can replicate some features of the complex architecture of mammalian microvasculature, but current processes fail to extend into the macro-scale. Structures which have features spanning multiple length scales are currently only fabricated through biological mechanisms and the relatively new field of biofabrication has developed, with the goal of utilizing and manipulating these processes.
Bioengineered tissues have been fabricated through a number of schemes, including direct printing and biospraying, in which cells and a supporting matrix are simultaneously deposited to form a complex network. See Mironov, V. et al. Organ printing: Tissue spheroids as building blocks. Biomaterials 30, 2164-2174, doi:10.1016/j.biomaterials.2008.12.084 (2009); Nakamura, M. et al. Biocompatible inkjet printing technique for designed seeding of individual living cells. Tissue Engineering 11, 1658-1666 (2005); and Campbell, P. G. & Weiss, L. E. Tissue engineering with the aid of inkjet printers. Expert Opin. Biol. Ther. 7, 1123-1127, doi:10.1517/14712598.7.8.1123 (2007). Centrifugal forces have been employed to create cross-linked hydrogels, with dense embedded cellular networks in tubular structures. Kasyanov, V. A. et al. Rapid biofabrication of tubular tissue constructs by centrifugal casting in a decellularized natural scaffold with laser-machined micropores. J. Mater. Sci.-Mater. Med. 20, 329-337, doi:10.1007/s10856-008-3590-3 (2009). Dielectrophoretic (Albrecht, D. R., Sah, R. L. & Bhatia, S. N. Geometric and material determinants of patterning efficiency by dielectrophoresis. Biophys. J. 87, 2131-2147, doi:10.1529/biophysj.104.039511 (2004)) and magnetic forces (Mironov, V., Kasyanov, V. & Markwald, R. R. Nanotechnology in vascular tissue engineering: from nanoscaffolding towards rapid vessel biofabrication. Trends Biotechnol. 26, 338-344, doi:10.1016/j.tibtech.2008.03.001 (2008)) have been employed to guide the arrangement of cells within synthetic matrices and cells embedded in a bio-polymer have been electrospun into tissue constructs (Stankus, J. J., Guan, J. J., Fujimoto, K. & Wagner, W. R. Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 27, 735-744, doi:10.1016/j.biomaterials.2005.06.020 (2006)). These techniques attempt to distribute cells within a suitable synthetically-fabricated network, leaving the embedded cells to reorganize into an optimal structure.
Decellularization of existing tissues extends the concept of biofabrication by taking advantage of the body's natural programming to create a complete tissue, including a functional vascular network. Rat liver extracellular matrix constructs have been created using chemical decellularization and reseeding. Baptista, P. M. et al. Generation of a Three-Dimensional Liver Bioscaffold with an Intact Vascular Network for Whole Organ Engineering (Wake Forest Institute for Regenerative Medicine Harvard-MIT Division of Health, Science, and Technology Rice University, 2009). Decellularized rat hearts, reseeded with multiple cell types, can contract and have the ability to generate pumping pressures. Ott, H. C. et al. Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nature Medicine 14, 213-221, doi:10.1038/nm1684 (2008). Challenges to chemical decellularization techniques include the potential for detergents to damage ECM components, the potential to create and deposit toxins, and the inherent difficulty of scaling these techniques up from small rat organs to larger organs. These challenges must be overcome before decellularized organs can successfully be translated to the clinical setting.
Xenotransplantation, or the transplantation of animal organs, is one potential solution to future organ shortages. Keeffe, E. B. Liver transplantation: Current status and novel approaches to liver replacement. Gastroenterology 120, 749-762, doi:10.1053/gast.2001.22583 (2001). Porcine xenotransplants have shown considerable potential, but have failed to become widely accepted or used. Transplantation of porcine pancreatic islets has recently been shown to temporarily reverse diabetes mellitus (See Cardona, K. et al. Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways. Nature Medicine 12, 304-306, doi:10.1038/nm1375 (2006); and Hering, B. J. et al. Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nature Medicine 12, 301-303, doi:10.1038/nm1369 (2006)) and the use of T-cell tolerance protocols have demonstrated the potential for long-term kidney transplantation in nonhuman primates. Yamada, K. et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha 1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nature Medicine 11, 32-34, doi:10.1038/nm1172 (2005).
Additionally, porcine livers have demonstrated the ability to clear ammonium and restore coagulation while under short term perfusion of human plasma. Chari, R. S. et al. Brief Report—Treatment of Hepatic Failure with ex-vivo Pig Liver Perfusion Followed by Liver Transplantation, N. Engl. J. Med. 331, 234-237 (1994); and Makowka, L. et al., The Use of a Pig Liver Zenograft for Temporary Support of a Patient with Fulminant Hepatic Failure, Transplantation 59, 1654-1659 (1995). Unfortunately, the mechanisms of graft loss and rejection in these transplants are still not well understood, and immunological rejection remains the most significant barrier to successful transplantation. Yang, Y. G. & Sykes, M. Xenotransplantation: current status and a perspective on the future. Nat. Rev. Immunol. 7, 519-531, doi:10.1038/nri2099 (2007).
Tissue engineering holds great promise for treating some of the most devastating diseases of our time. Because engineered tissue and organ replacements can be developed in a laboratory, therapies can potentially be delivered on a large scale, for multiple disease states with dramatic reduction in waiting times for patients. The concept of engineering tissue using selective cell transplantation has been applied experimentally and clinically for a variety of disorders, including the successful use of engineered bladder tissue for bladder reconstruction, engineered injectable chondrocytes for the treatment of vesicoureteral reflux and urinary incontinence, and vascular grafts. For clinical use for humans, the process involves the in vitro seeding and attachment of human cells onto a scaffold. Once seeded, the cells proliferate, migrate into the scaffold, and differentiate into the appropriate cell type for the specific tissue of interest while secreting the extracellular matrix components required to create the tissue. The three dimensional structure of the scaffold, and in particular the size of pores and density of the scaffold, is important in successful proliferation and migration of seeded cells to create the tissue of interest. Therefore, the choice of scaffold is crucial to enable the cells to behave in the required manner to produce tissues and organs of the desired shape and size.
To date, scaffolding for tissue engineering has usually consisted of natural and synthetic polymers. Methods known in the art for forming scaffolds for tissue engineering from polymers include solvent-casting, particulate-leaching, gas foaming of polymers, phase separation, and solution casting. Electrospinning is another popular method for creating scaffolds for engineered tissues and organs, but widely used techniques suffer from fundamental manufacturing limitations that have, to date, prevented its clinical translation. These limitations result from the distinct lack of processes capable of creating electrospun structures on the nano-, micro-, and millimeter scales that adequately promote cell growth and function.
Of fundamental importance to the survival of most engineered tissue scaffolds is gas and nutrient exchange. In nature, this is accomplished by virtue of microcirculation, which is the feeding of oxygen and nutrients to tissues and removing waste at the capillary level. However, gas exchange in most engineered tissue scaffolds is typically accomplished passively by diffusion (generally over distances less than 1 mm), or actively by elution of oxygen from specific types of material fibers. Microcirculation is difficult to engineer, particularly because the cross-sectional dimension of a capillary is only about 5 to 10 micrometers (μm; microns) in diameter. As yet, the manufacturing processes for engineering tissue scaffolds have not been developed and are not capable of creating a network of blood vessels. Currently, there are no known tissue engineering scaffolds with a circulation designed into the structure for gas exchange. As a result, the scaffolds for tissues and organs are limited in size and shape.
In addition to gas exchange, engineered tissue scaffolds must exhibit mechanical properties comparable to the native tissues that they are intended to replace. This is true because the cells that populate native tissues sense physiologic strains, which can help to control tissue growth and function. Most natural hard tissues and soft tissues are elastic or viscoelastic and can, under normal operating conditions, reversibly recover the strains to which they are subjected. Accordingly, engineered tissue constructs possessing the same mechanical properties as the mature extracellular matrix of the native tissue are desirable at the time of implantation into the host, especially load bearing structures like bone, cartilage, or blood vessels.
There are numerous physical, chemical, and enzymatic ways known in the art for preparing scaffolds from natural tissues. Among the most common physical methods for preparing scaffolds are snap freezing, mechanical force (e.g., direct pressure), and mechanical agitation (e.g., sonication). Among the most common chemical methods for preparing scaffolds are alkaline or base treatment, use of non-ionic, ionic, or zwitterionic detergents, use of hypo- or hypertonic solutions, and use of chelating agents. Common enzymatic methods for preparing scaffolds include the use of trypsin, endonucleases, or exonucleases. Currently, it is recognized in the art that, to fully decellularize a tissue to produce a scaffold, two or more of the above-noted ways, and specifically two or more ways from different general classes (i.e., physical, chemical, enzymatic), should be used. Unfortunately, the methods used must be relatively harsh on the tissue so that complete removal of cellular material can be achieved. The harsh treatments invariable degrade the resulting scaffold, destroying vasculature and neural structures.
The most successful scaffolds used in both pre-clinical animal studies and in human clinical applications are biological (natural) and made by decellularizing organs of large animals (e.g., pigs). In general, removal of cells from a tissue or an organ for preparation of a scaffold should leave the complex mixture of structural and functional proteins that constitute the extracellular matrix (ECM). The tissues from which the ECM is harvested, the species of origin, the decellularization methods and the methods of terminal sterilization for these biologic scaffolds vary widely. However, as mentioned above, the decellularization methods are relatively harsh and result in significant destruction or degradation of the extracellular scaffold. Once the scaffold is prepared, human cells are seeded so they can proliferate, migrate, and differentiate into the specific tissue. The intent of most decellularization processes is to minimize the disruption to the underlying scaffold and thus retain native mechanical properties and biologic properties of the tissue. However, to date this intent has not been achieved. Snap freezing has been used frequently for decellularization of tendinous, ligamentous, and nerve tissue. By rapidly freezing a tissue, intracellular ice crystals form that disrupt cellular membranes and cause cell lysis. The rate of temperature change must be carefully controlled to prevent the ice formation from disrupting the ECM as well. While freezing can be an effective method of cell lysis, it must be followed by processes to remove the cellular material from the tissue.
Cells can be lysed by applying direct pressure to tissue, but this method is only effective for tissues or organs that are not characterized by densely organized ECM (e.g., liver, lung). Mechanical force has also been used to delaminate layers of tissue from organs that are characterized by natural planes of dissection, such as the small intestine and the urinary bladder. These methods are effective, and cause minimal disruption to the three-dimensional architecture of the ECM within these tissues. Furthermore, mechanical agitation and sonication have been utilized simultaneously with chemical treatment to assist in cell lysis and removal of cellular debris. Mechanical agitation can be applied by using a magnetic stir plate, an orbital shaker, or a low profile roller. There have been no studies performed to determine the optimal magnitude or frequency of sonication for disruption of cells, but a standard ultrasonic cleaner appears to be effective. As noted above, currently used physical treatments are generally insufficient to achieve complete decellularization, and must be combined with a secondary treatment, typically a chemical treatment. Enzymatic treatments, such as trypsin, and chemical treatment, such as ionic solutions and detergents, disrupt cell membranes and the bonds responsible for intercellular and extracellular connections. Therefore, they are often used as a second step in decellularization, after gross disruption by mechanical means.
Although advances have been made recently in the field of IRE and the concept of treatment of tumors with IRE has been established, the present inventors have recognized that there still exists a need in the art for improved devices and methods for ablating diseased tissues using IRE. More specifically, the inventors employ the vascular bed of tissues as a physiologic electrode (“Physiologic Vascular Electrode” or “PVE”) to selectively ablate cells in soft tissue. Application of this technique ex vivo and in vivo to ablate unwanted cells can be useful for treatment of aggressive, infiltrative and circumscribed neoplasms, as well as a wide variety of applications in tissue engineering, tissue regeneration, and organ transplantation employing biologically-derived tissue constructs.